In the year 2002, the human genome sequence has been decoded almost completely. From the obtained data of bases, the developments of tailor-made therapies and so on have been expected. For this purpose, it is very important to identify genes and determine the degree of their expression in vivo, or to elucidate the network of gene expression. In addition, for elucidating the gene-expression network, there is a need for making a gene expression profile, which identifies genes being expressed and determines the degree of their expression in vivo at a specified time.
At present, methods for preparing a gene expression profile include a differential display method, a serial analysis of gene expression (SAGE), a method using a micro-array or a DNA chip, a method of high-coverage expression profiling (HiCEP) disclosed in the International Publication WO 02/48352 A1, etc.
Among these methods for preparing a gene expression profile, the HICEP method is an excellent one in that the gene expression profile can be easily prepared for covering a wide variety of genes including unknown genes.
In a PCR reaction used as one of the steps in the HiCEP method, the temperature used for annealing a primer to a template is conventionally around the melting temperature (Tm) of the primer, for example, 55° C. when each primer used was composed of a 20 mer with a 50% GC content (“Protocol 15: Quantitative PCR 8.86” in Sambrook and Russell, “Molecular Cloning”, 3rd Ed., Cold Spring Harbor Laboratory Press (U.S.A.), 2001).
The expression level of a gene varies depending on the kinds of genes and the expression timing thereof. Therefore, for elucidating the gene expression network, a method for preparing a gene expression profile, as a prerequisite thereof, is desired to have high detection sensitivity to expressed genes.
Accordingly, for a HiCEP method capable of preparing a gene expression profile covering a wide variety of genes in a simple manner, the present inventor has intensively studied for a further improvement in the sensitivity of detecting expressed genes.